Blackleg or stem canker is a major disease of Brassica napus L. (oilseed rape or Canola), causing annually major economic losses worldwide, in particular in Europe, Australia and North America. Blackleg is caused by the fungal pathogen Leptosphaeria maculans (Desm.) Ces. & De Not. (anamorph Phoma lingam Tode ex. Fr.). L. maculans symptoms can develop on cotyledons, leaves, pods and stems. Leaf lesions develop after infection by wind dispersed ascospores and/or water (splash) dispersed conidiospores. Stem symptoms (or cankers) can arise through direct infection of the stems or through systemic growth of the fungus from leaf lesions, through the vascular tissue into the stem [Hammond et al. (1985), Plant Pathology 34: 557-565]. Stem cankers may girdle the stem, which can lead to the lodging of plants and plant death. Less severe cankers can cause a restriction in water and nutrient flow, which in turn may lead to shriveling of seeds and pods. Pod infection can lead to premature podshatter and seed infection.
The incorporation of blackleg resistance into B. napus cultivars is one of the major objectives in breeding programs worldwide. Although both the spraying of fungicides and cultural practices are used to reduce yield losses caused by blackleg infection, the most reliable method of control to date is genetic resistance. Brassica napus (2n=38, genome AACC) is an amphidiploid species, which originated from a spontaneous hybridization of Brassica rapa L. (syn. B. campestris; 2n=20, AA) and Brassica oleracea L. (2n=18, CC). B. napus contains the complete chromosome sets of these two diploid genomes.
Blackleg resistance is assessed either in glasshouse or in field experiments, in one embodiment blackleg resistance is preferably assessed in field experiments, and can be assessed at different stages of the plant development. When referring to blackleg resistance, normally different types of resistance are therefore distinguished depending on the plant stage and tissue assessed, such as seedling resistance (‘early’ resistance) and adult plant resistance (‘late’ or ‘stem’ resistance). Plant tissues analyzed for resistance are for example cotyledons, leaves and stem bases. Genetical resistance to blackleg has been reported to be either monogenic (under control of a major gene) or polygenic (under control of several minor genes).
A number of resistance loci have been mapped in B. napus. For example, a single dominant resistance locus, designated LEM1, was reported to be located on linkage group 6 (which is now known to the inventors to be chromosome N07 in the nomenclature of Sharpe et al. (1995, Genome 38: 1112-1121)) of B. napus cv. Major, based on wound inoculations of seedlings [Fereirra et al. (1995), Genetics 85 (2): 213-217]. Field resistance in adult plants, in spring cv. Cresor, was mapped to chromosome N07 by Dion et al. [(1995), TAG 91: 1190-1194] and designated LmFr. Cultivars Maluka and Shiralee were reported to have a major locus controlling seedling resistance, designated LmR1, on chromosome N07 [Mayerhofer et al. (1997), Genome 40: 294-301.]. Rimmer et al. [(1999), Proceedings of the 10th International Rapeseed Congress] also reported resistance loci, designated RLM, on chromosome N07.
However, the lack of adequate resistance found in Brassica napus (AACC genome) and the continuous threat of breakdown of resistance when a resistant cultivar is used widespread and over longer time periods, has lead breeders and scientists to search for alternative sources of resistance. The main focus has been on the identification and transfer of resistance alleles from related Brassica species, such as B. rapa (AA), B. oleracea (CC), B. nigra (BB genome), B. juncea (AABB genome) and B. carinata (BBCC).
One major source of blackleg resistance is the B genome. Gerdemann-Knörck reported in 1994 the introduction of blackleg resistance into B. napus from B. nigra by asymmetric somatic hybridization [Gerdemann-Knörck et al. (1994), Plant Breeding 113: 106-113].
Another approach has been to generate so-called ‘synthetic’ B. napus lines by interspecific hybridization of two diploid species (AA and CC genome) and subsequent in vitro culture of embryos and chromosome doubling.
Blackleg resistance was introduced into B. napus in this way by generating synthetic B. napus plants from wild B. rapa (AA genome) accessions [Crouch et al. (1994), Plant Breeding 112: 265-278] and wild B. atlantica (CC genome) accessions [Mithen and Magrath (1992), Plant Breeding 108: 60-68, and Mithen and Herron (1991), Proceedings of the 8th International Rapeseed Congress]. 
Six accessions of wild B. rapa ssp sylvestris were crossed with B. oleracea ssp alboglabra in order to develop a series of synthetic B. napus lines with a common C genome but different A genomes [Crouch et al. (1994), supra]. B. rapa ssp sylvestris #75 and #76 were found to be resistant to blackleg isolates in glasshouse tests (cotyledon and leaf tests), while B. rapa ssp sylvestris #29 was susceptible. Two of the synthetic lines derived from #75 or #76 and B. oleracea ssp alboglabra, and their F1 hybrids with oilseed rape cultivars, showed high resistance to blackleg in glasshouse experiments. Only one of these lines also showed resistance in field experiments in England and Australia.
Crouch (PhD thesis, University of East Anglia, Norwich, UK) describes RFLP markers linked to regions of the genome contributing to field resistance, the mapping of these regions to five linkage groups and the localization of quantitative trait loci (QTL) contributing to resistance in different tissues. Interval analysis identified QTL contributing to leaf resistance in both Group 1 from the synthetic parent [chromosome N7 according to Sharpe et al. (1995), Genome 38: 1112-1121] and Group 3 from the cultivar parent (chromosome N3 according to Sharpe et al.) and QTL contributing to resistance in the lower part of the stem, hypocotyl and root on Group 1, Group 2 (chromosome N10 according to Sharpe et al.) and Group 5 (the association of this group with the linkage groups of Sharpe et al., is uncertain). Interval mapping failed to identify any QTL contributing to resistance in the upper part of the stem.
The synthetic B. napus lines described by Crouch and Mithen (supra) were agronomically not suitable, as they contained high glucosinolate levels, high erucic acid levels, had poor fertility and suffered from self-incompatibility [Easton, Australian Research Assembly on Brassicas (2001)].
In spring 2000 Pacific Seeds brought the open-pollinated B. napus variety Surpass400 onto the market in Australia, which received a national blackleg resistance rating of 9.0, the highest known level of resistance. The ancestry of Surpass400 includes a ‘synthetic’ B. napus, derived from interspecific crosses between wild B. rapa ssp sylvestris from Sicily and B. oleracea ssp alboglabra [Li et al., Australian Research Assembly on Brassicas 2001; Easton, supra]. A major dominant allele for blackleg resistance at the seedling stage was reported to be present in Surpass400 [Li et al., Australian Research Assembly on Brassicas 2001].
Yu et al. [(2002), Can. J. Plant Pathology 24: 96-97; Plant, Animal & Microbe Genomes Conference Jan. 12-16, (2002)] reported two resistance loci in B. napus populations derived from crosses with breeding lines 6270 and 6279. The dominant nuclear allele designated LepR1 on chromosome NO2 conferred resistance in line 6270 to L. maculans isolates from pathogenicity groups PG2, PG3 and PG4. The second locus, designated LepR2 located on chromosome N10, was incompletely dominant and conferred cotyledon resistance to PG2 and PG3 isolates.
Rimmer et al. [13th Crucifer Genetics Workshop, Mar. 23-26, (2002)] reported the mapping of four resistance loci in B. napus. Two resistance loci were derived from B. napus (not from B. rapa) and mapped to chromosome N7 and chromosome N8. The other two loci were derived from B. rapa ssp sylvestris and mapped to chromosome 2 and chromosome 10.
Early 2003 the first reports of a breakdown of Surpass400 resistance were made. A more virulent strain of the fungus seems to have evolved in just three years, able to infect Surpass400. How quickly this strain will be able to spread to different locations remains to be seen, but new resistance genes and methods of enhancing durability of resistance are clearly needed.
With the constant threat of genetic resistance breaking down as a result of changes in the pathogen population, it is desirable to identify new genetic sources of resistance, methods for transferring these into varieties with high agronomic performance and methods for enhancing durability of resistance. The present invention, including the different embodiments provided in the specifications and claims, provides plants comprising a novel blackleg resistance gene, Lem-08-syl and methods and means for transferring Lem-08-syl into other breeding lines or varieties, as well as methods of detecting the presence/absence of Lem-08-syl in plants.